Potent carbonic anhydrase I, II, IX and XII inhibition activity of novel primary benzenesulfonamides incorporating bis-ureido moieties

Abstract A novel series of twelve aromatic bis-ureido-substituted benzenesulfonamides was synthesised by conjugation of aromatic aminobenzenesulfonamides with aromatic bis-isocyanates. The obtained bis-ureido-substituted derivatives were tested against four selected human carbonic anhydrase isoforms (hCA I, hCA II, hCA IX and hCA XII). Most of the new compounds showed an effective inhibitory profile against isoforms hCA IX and hCA XII, also having some selectivity with respect to hCA I and hCA II. The inhibition constants of these compounds against isoforms hCA IX and XII were in the range of 6.73–835 and 5.02–429 nM, respectively. Since hCA IX and hCA XII are important drug targets for anti-cancer/anti-metastatic drugs, these effective inhibitors reported here may be considered of interest for cancer related studies in which these enzymes are involved.

Ureido-substituted primary benzenesulfonamides were extensively studied as potent and selective CA inhibitors (CAIs), and among them 4-[[(4-fluorophenyl) carbamoyl] amino] benzenesulfonamide (SLC-0111) ( Figure 1) has reached to Phase Ib/II clinical trials for the management of advanced metastatic solid tumors [12][13][14] . Initially, ureido-substituted benzenesulfonamides were investigated as hCA I, hCA II, and bCA IV inhibitors possessing high activity and unexpectedly high selectivity 25 . Later, other such ureido bearing compounds (benzenesulfonamides and benzenesulfamates) have been designed, synthesised and explored as potent and selective hCA IX and XII inhibitors [12][13][14] . On the other hand, more recently, secondary and tertiary benzenesulfonamides started to be also investigated as CAIs, some of which showed strong affinity against several important isozymes, although they were generally less effective, compared to primary counterparts 26,27 .
In continuation of our recent efforts to develop effective and isoform selective CAIs [28][29][30] , in the present study, we report bis-ureido-substituted primary benzenesulfonamides acting as potent human (h) hCA inhibitors using the design strategy summarised in Figure 1. To the best of our knowledge, this is the first bis-ureidosubstituted primary benzenesulfonamide study, which examined the inhibition profile of these compounds on selected hCAs, namely off-target cytosolic isoforms hCA I and II, and tumouroverexpressed membrane-bound isoforms hCA IX and XII.

Chemistry
Unless otherwise noted, all the chemicals and anhydrous solvents were purchased from Sigma-Aldrich, Merck, Alfa Aesar and TCI and used without further purification. Melting points (mp) were determined with SMP20 melting point apparatus and are uncorrected. FT-IR spectra were obtained by using Perkin Elmer Spectrum 100 FT-IR spectrometer. Nuclear Magnetic Resonance ( 1 H-NMR and 13 C-NMR) spectra of compounds were recorded using a Bruker Advance III 300 Mhz spectrometer in DMSO-d 6 as the solvent, and TMS as the internal standard operating at 300 Mhz for 1 H-NMR and 75 Mhz for 13 C-NMR. Chemical shifts are expressed in ppm relative to tetramethylsilane. Splitting patterns are designated as singlet (s), doublet (d), triplet (t), quartette (q), and multiplet (m). Thin layer chromatography (TLC) was carried out on Merck silica gel 60 F 254 plates.

CA inhibition
An SX.18 MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay the catalytic/inhibition of various CA isozymes 31 . Phenol Red (at a concentration of 0.2 mM) has been used as an indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.4) as a buffer, 0.1 M Na 2 SO 4 or NaClO 4 (for maintaining constant the ionic strength; these anions are not inhibitory in the used concentration), following the CA-catalyzed CO 2 hydration reaction for a period of 5-10 s. Saturated CO 2 solutions in water at 25 C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of 10 mM (in DMSO-water 1:1, v/v) and dilutions up to 0.01 nM done with the assay buffer mentioned above. At least 7 different inhibitor concentrations have been used for measuring the inhibition constant. Inhibitor and enzyme solutions were pre-incubated together for 10 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. Triplicate experiments were done for each inhibitor concentration, and the values reported throughout the paper is the mean of such results. The inhibition constants were obtained by nonlinear least-squares methods using the Cheng-Prusoff equation, as reported earlier [32][33][34][35][36][37] , and represent the mean from at least three different determinations [38][39][40][41][42][43][44][45] . All CA isozymes used here were recombinant proteins obtained as reported earlier by our group and their concentrations were in the range of 6-14 nM 38 -45.

Results and discussion
General synthesis and design strategy of the compounds In recent studies, CAIs having bifunctional pharmacophores in their structures were reported, in order to improve biological activity and selectivity of compounds by ditopic interactions on the active site of the same or different enzyme(s) [46][47][48][49][50] . In the context of the bis-substituted CAI design approach, we focussed on the development of a new bis-ureido-substituted benzenesulfonamides. Our aim was to produce molecules which have two binding groups in their structure and the ureido linker in between them to investigate isoform selectivity and potency of compounds by a potentially synergistic/multivalent effect. On the other hand, we aimed to find the relationship between the effect of the zinc binding group (primary sulphonamide part) and the linker position and length (ureido part) against various CA isozyme of pharmacologic relevance. To achieve this, we have used three well known primary benzenesulfonamide pharmacophores namely, sulphanilamide, metanilamide and 4-(2-aminoethyl)benzenesulfonamide which have been converted to aromatic bis-ureido derivatives (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Hence, these primary benzenesulfonamide derivatives were condensed with four different aromatic bis-isocyanate moieties under mild conditions to produce twelve novel bis-ureido-substituted benzenesulfonamides derivatives. The general synthetic route is shown in the Scheme 1. Briefly, acetonitrile was used as a solvent and the reaction temperature was from room temperature to 50 C, overnight. Table 1. Inhibition data of human CA isoforms hCA I, hCA II, hCA IX and hCA XII with bis-ureido-substituted primary benzenesulfonamide derivatives (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) reported here and standard sulphonamide inhibitor Acetazolamide (AAZ) by a stopped flow CO 2 hydrase assay.

Carbonic anhydrase inhibition
All the newly synthesised bis-ureido-substituted primary benzenesulfonamide derivatives were evaluated for their CA inhibition properties against two cytosolic off-target isoforms (hCA I and II) and two membrane-bound isoforms (hCA IX and XII) by using a stopped-flow assay.The well-known CAI drug acetazolamide (AAZ) was used as a standard for comparison, and all the obtained results are summarised in Table 1

Conclusions
A series of novel primary benzenesulfonamides incorporating bisureido moieties were synthesised through the conjugation of sulphonamides with aromatic bis-isocyanates under mild conditions using SLC-0111 as lead compound. These bifunctional CAI design strategy was applied to obtain potent and possibly selective inhibitors incorporating the ureido linker. The inhibition properties of the obtained compounds were evaluated against four selected CA isoforms, including cytosolic ones (hCA I and II), as well as the tumour-associated membrane-bound isoforms (hCA IX and XII). In general, these compounds showed weak hCA I inhibition with K i s ranging from 68.1 to 9174 nM and effective to moderate inhibition against another cytosolic isoform hCA II with K i s of 4.4 to 792 nM. The tumour-associated membrane-bound isoforms hCA IX and XII were effectively inhibited by some of these compounds with K i s in the range of 6.73-835 and 5.02-429 nM, respectively. Interestingly, compounds 11 and 19 were found to be potent derivatives against these isoforms in which both of them have the same ureido linker (1,3-substituted phenyl ring) in their structure. However, compound 19 was more selective against the off-target isoforms hCA I and II. As a result, the potent inhibition properties of these novel compounds against tumour-associated isoforms hCA IX and XII making these bis-ureido substituted compounds of interest for antimetastatic drug design research.

Acknowledgement
TT is thankful to Gazi University for the support of her PhD studies. The authors are grateful to Gazi University BAP [Gazi University Scientific Research Projects Coordination Department grant no: FDK-2022-7551] for the financial support of the current work.

Disclosure statement
All authors except CTS report no conflict of interest. CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.